金屬硫蛋白與血清白蛋白是生物體內能與重金屬結合的兩種主要蛋白質，在重金屬的傳送、儲存及解毒等功能上扮演著重要的角色。近年來，重金屬污染已成為一種全球性的危害，因此，一個快速、有效的偵測方法是迫切需要的。在本實驗中，我們嘗試建立蛋白質生物感應片偵測系統，配合表面電漿共振技術來偵測並定量重金屬離子。本實驗分兩個部分，第一部分我們先將金屬硫蛋白吸附在CM5感應片上，其最佳吸附能力之pH值為4。金屬硫蛋白感應片在30℃時，與金屬有較好的結合情形，而流速則對結合力沒有影響。此外，感應片對不同金屬也有區別性，與鎘、鋅和鎳等金屬具結合能力，但對錳、鎂和鈣則沒有。而鎘、鋅和鎳等金屬在不同濃度範圍內與金屬硫蛋白感應片的結合能力有很好的相關性，其中，鎘的偵測極限可達1 □M。另外，緩衝溶液中的NaCl、pH值及 Tween 20等成分都會影響金屬和金屬硫蛋白感應片的結合情形，當NaCl濃度將低至1 mM時，只能和鎘結合，而無法與鋅和鎳結合。此外，在分子之間的結合動力學研究中發現金屬硫蛋白感應片對金屬的親和力為鎘＞鋅＞鎳。因此金屬硫蛋白感應片不但可以評估和金屬結合的特性，也適合發展成為偵測及定量重金屬的生物感應器。
在第二部份，我們改將血清白蛋白吸附在CM5感應片上，其最佳吸附能力之pH值為5。系統的溫度及緩衝溶液中的成分都會影響金屬和感應片的結合情形。在25℃、1 mM NaCl環境中，感應片與鎘、鋅和鎳等金屬有最佳結合能力，而與錳、鎂和鈣結合能力較差。血清白蛋白感應片可以偵測不同濃度範圍的鎘、鋅和鎳等金屬，其濃度與結合能力有很好的相關性。其中，對鎘、鋅和鎳等金屬的偵測極限可達0.01 uM∼0.1 uM。在競爭實驗中，我們發現鋅和鎳會降低20∼30％鎘的結合能力，而鋅和鎳彼此則無競爭現象發生。同時，在動力學研究發現血清白蛋白感應片對金屬的親和力為鎳＞鋅＞鎘。此外，血清白蛋白感應片也可以經由簡單的管柱分離而順利的偵測到血清樣品中鎘的濃度。因此血清白蛋白感應片除了可以評估和金屬結合的特性，也適合發展成為偵測及定量溶液中及生物體內重金屬的生物感應器。

Metallothionein (MT) and serum albumin are two important metal-binding proteins, play roles in metal transport, storage and detoxification in organism. In this study, we attempt to establish protein-based biosensors for the detection of metal ions by surface plasmon resonance. In the first part, rabbit MT was immobilized onto a carboxymethylated dextran matrix (CM5 chip). The optimal pH for the immobilization was determined at 4, and the highest interaction temperature was observed at 30oC. The sensor chip binds cadmium, zinc or nickel, but not magnesium, manganese and calcium. Calibration curves for the quantification of metal ions showed excellent linearity. The sensitivity for metal detection was at the micromolar level. The interaction between the metal ions and the sensor chip was significantly influenced by the presence of NaCl, Tween 20 and the pH of the reaction buffer. In the environment containing 1 mM NaCl, the MT chip effectively differentiated cadmium from zinc and nickel. In displacement analysis, zinc and nickel did not affect cadmium-MT interaction. The binding affinity between the metal ions and the immobilized MT follows the order of cadmium > zinc > nickel.
In the second part, bovine serum albumin was immobilized onto a CM5 chip. The optimal pH for the immobilization was determined at 5, and the highest interaction temperature was found at 25oC. The interaction between the metal ions and the sensor chip was significantly influenced by the reaction buffer. The optimal buffer condition used for the analysis contains 1 mM NaCl, 0.005% Tween-20 and 0.01 M HEPES, pH 7.4. Using this condition, a linear calibration curve can be established within the range of 10-8 to 10-4 M for metals. The sensitivity for cadmium, zinc and nickel detection were at the 0.01~0.1 uM. When measuring the solution containing two species of metal ions by the albumin chip, zinc and nickel were able to reduce cadmium-albumin interaction about 20~30%. However, zinc and nickel show an additive in binding to the chip when both metals are mixed together. The binding affinity between the metal ions and the immobilized albumin follows the order of nickel > zinc > cadmium. A procedure was also developed to analyze cadmium content in the serum using the albumin-based sensor chip. Results from our studies show that the protein-based biosensor can be effectively used for the detection and measurement of metal ions in the solution and in biological sample.

Alvarez De Eulate MJ, Montoro R, Ybanez N, De La Guardia M. 1986. Determination of cadmium, copper, and lead in sodium chloride food salts by flame atomic absorption spectroscopy. J Assoc Off Anal Chem 69(5):871-3.
Anderson. J. L. B, E. F., Pickup, P. G. 1996. Dynamic electrochemistry: methodology and application. Anal Chem 68:379R-444R.
Angerer JaS, K.H. 1985. Analyses of hazardous substance in biological materials.
Arseniev A, Schultze P, Worgotter E, Braun W, Wagner G, Vasak M, Kagi JH, Wuthrich K. 1988. Three-dimensional structure of rabbit liver [Cd7]metallothionein-2a in aqueous solution determined by nuclear magnetic resonance. J Mol Biol 201(3):637-57.
Bader B, Kuhn K, Owen DJ, Waldmann H, Wittinghofer A, Kuhlmann J. 2000. Bioorganic synthesis of lipid-modified proteins for the study of signal transduction. Nature 403(6766):223-6.
Bontidean I, Ahlqvist J, Mulchandani A, Chen W, Bae W, Mehra RK, Mortari A, Csoregi E. 2003. Novel synthetic phytochelatin-based capacitive biosensor for heavy metal ion detection. Biosens Bioelectron 18(5-6):547-53.
Bontidean I, Berggren C, Johansson G, Csoregi E, Mattiasson B, Lloyd JR, Jakeman KJ, Brown NL. 1998. Detection of heavy metal ions at femtomolar levels using protein-based biosensors. Anal Chem 70(19):4162-9.
Braun W, Vasak M, Robbins AH, Stout CD, Wagner G, Kagi JH, Wuthrich K. 1992. Comparison of the NMR solution structure and the x-ray crystal structure of rat metallothionein-2. Proc Natl Acad Sci U S A 89(21):10124-8.
Burger J, Lord CG, Yurkow EJ, McGrath L, Gaines KF, Brisbin IL, Jr., Gochfeld M. 2000. Metals and metallothionein in the liver of raccoons: utility for environmental assessment and monitoring. J Toxicol Environ Health A 60(4):243-61.
Dabeka RW. 1989. Graphite-furnace atomic absorption spectrometric determination of lead, cadmium, cobalt and nickel in infant formulas and evaporated milks after nitric-perchloric acid digestion and coprecipitation with ammonium pyrrolidine dithiocarbamate. Sci Total Environ 89(3):271-7.
Doz F, Roosen N, Rosenblum ML. 1993. Metallothionein and anticancer agents: the role of metallothionein in cancer chemotherapy. J Neurooncol 17(2):123-9.
Dudley RE, Svoboda DJ, Klaassen C. 1982. Acute exposure to cadmium causes severe liver injury in rats. Toxicol Appl Pharmacol 65(2):302-13.
Durnam DM, Palmiter RD. 1984. Induction of metallothionein-I mRNA in cultured cells by heavy metals and iodoacetate: evidence for gratuitous inducers. Mol Cell Biol 4(3):484-91.
Fisher RJ, Fivash M. 1994. Surface plasmon resonance based methods for measuring the kinetics and binding affinities of biomolecular interactions. Curr Opin Biotechnol 5(4):389-95.
Fowler BA, Hildebrand CE, Kojima Y, Webb M. 1987. Nomenclature of metallothionein. Experientia Suppl 52:19-22.
Goering PL, Klaassen CD. 1984. Resistance to cadmium-induced hepatotoxicity in immature rats. Toxicol Appl Pharmacol 74(3):321-9.
Hamer DH. 1986. Metallothionein. Annu Rev Biochem 55:913-51.
Hechtenberg S, and Beyersmann, D. 1995. Regulation of nuclear calcium and zinc -Interference by toxic metal ions . In: metals. Igrt, editor. New Youk.
Jackson PJ, Unkefer CJ, Doolen JA, Watt K, Robinson NJ. 1987. Poly(gamma-glutamylcysteinyl)glycine: its role in cadmium resistance in plant cells. Proc Natl Acad Sci U S A 84(19):6619-23.
Jiang LJ, Vasak M, Vallee BL, Maret W. 2000. Zinc transfer potentials of the alpha - and beta-clusters of metallothionein are affected by domain interactions in the whole molecule. Proc Natl Acad Sci U S A 97(6):2503-8.
Kagi JH. 1991. Overview of metallothionein. Methods Enzymol 205:613-26.
Kagi JH, Schaffer A. 1988. Biochemistry of metallothionein. Biochemistry 27(23):8509-15.
Kagi JH, Valee BL. 1960. Metallothionein: a cadmium- and zinc-containing protein from equine renal cortex. J Biol Chem 235:3460-5.
Kagi JH, HaK Y. 1987. Chemistry and biochemistry of metallothionein. Experientia Suppl 52:25-61.
Karlsson R, Falt A. 1997. Experimental design for kinetic analysis of protein-protein interactions with surface plasmon resonance biosensors. J Immunol Methods 200(1-2):121-33.
Knibiehler M, Goubin F, Escalas N, Jonsson ZO, Mazarguil H, Hubscher U, Ducommun B. 1996. Interaction studies between the p21Cip1/Waf1 cyclin-dependent kinase inhibitor and proliferating cell nuclear antigen (PCNA) by surface plasmon resonance. FEBS Lett 391(1-2):66-70.
Kuswandi B. 2003. Simple optical fibre biosensor based on immobilised enzyme for monitoring of trace heavy metal ions. Anal Bioanal Chem 376(7):1104-10.
Lehman-McKeeman LD, Andrews GK, Klaassen CD. 1988. Ontogeny and induction of hepatic isometallothioneins in immature rats. Toxicol Appl Pharmacol 92(1):10-7.
Liska SK, Kerkay J, Pearson KH. 1985. Determination of zinc and copper in urine using Zeeman effect flame atomic absorption spectroscopy. Clin Chim Acta 151(3):231-6.
Liu J, Kershaw WC, Klaassen CD. 1991. The protective effect of metallothionein on the toxicity of various metals in rat primary hepatocyte culture. Toxicol Appl Pharmacol 107(1):27-34.
Lopez-Artiguez M, Camean A, Repetto M. 1993. Preconcentration of heavy metals in urine and quantification by inductively coupled plasma atomic emission spectrometry. J Anal Toxicol 17(1):18-22.
Lu Y, Liu J, Li J, Bruesehoff PJ, Pavot CM, Brown AK. 2003. New highly sensitive and selective catalytic DNA biosensors for metal ions. Biosens Bioelectron 18(5-6):529-40.
Margoshe M, Vallee BL. 1957. A Cadmium protein from equine kidney cortex. J Am Chem Soc 79:4813-4814.
Masters SC, Pederson KJ, Zhang L, Barbieri JT, Fu H. 1999. Interaction of 14-3-3 with a nonphosphorylated protein ligand, exoenzyme S of Pseudomonas aeruginosa. Biochemistry 38(16):5216-21.
Menegario AA, Packer AP, Gine MF. 2001. Determination of Ba, Cd, Cu, Pb and Zn in saliva by isotope dilution direct injection inductively coupled plasma mass spectrometry. Analyst 126(8):1363-6.
Mernagh DR, Janscak P, Firman K, Kneale GG. 1998. Protein-protein and protein-DNA interactions in the type I restriction endonuclease R.EcoR124I. Biol Chem 379(4-5):497-503.
Mourgaud Y, Martinez E, Geffard A, Andral B, Stanisiere JY, Amiard JC. 2002. Metallothionein concentration in the mussel Mytilus galloprovincialis as a biomarker of response to metal contamination: validation in the field. Biomarkers 7(6):479-90.
Nartey NO, Banerjee D, Cherian MG. 1987. Immunohistochemical localization of metallothionein in cell nucleus and cytoplasm of fetal human liver and kidney and its changes during development. Pathology 19(3):233-8.
Nettesheim DG, Engeseth HR, Otvos JD. 1985. Products of metal exchange reactions of metallothionein. Biochemistry 24(24):6744-51.
Nielson KB, Atkin CL, Winge DR. 1985. Distinct metal-binding configurations in metallothionein. J Biol Chem 260(9):5342-50.
Nielson KB, Winge DR. 1983. Order of metal binding in metallothionein. J Biol Chem 258(21):13063-9.
Nielson KB, Winge DR. 1984. Preferential binding of copper to the beta domain of metallothionein. J Biol Chem 259(8):4941-6.
Quaife CJ, Findley SD, Erickson JC, Froelick GJ, Kelly EJ, Zambrowicz BP, Palmiter RD. 1994. Induction of a new metallothionein isoform (MT-IV) occurs during differentiation of stratified squamous epithelia. Biochemistry 33(23):7250-9.
Ramanathan S, Ensor M, Daunert S. 1997. Bacterial biosensors for monitoring toxic metals. Trends Biotechnol 15(12):500-6.
Rensing C, Maier RM. 2003. Issues underlying use of biosensors to measure metal bioavailability. Ecotoxicol Environ Saf 56(1):140-7.
Robbins AH, McRee DE, Williamson M, Collett SA, Xuong NH, Furey WF, Wang BC, Stout CD. 1991. Refined crystal structure of Cd, Zn metallothionein at 2.0 A resolution. J Mol Biol 221(4):1269-93.
Saber R, Piskin E. 2003. Investigation of complexation of immobilized metallothionein with Zn(II) and Cd(II) ions using piezoelectric crystals. Biosens Bioelectron 18(8):1039-46.
Stillman MJ, Cai W, Zelazowski AJ. 1987. Cadmium binding to metallothioneins. Domain specificity in reactions of alpha and beta fragments, apometallothionein, and zinc metallothionein with Cd2+. J Biol Chem 262(10):4538-48.
Thornalley PJ, Vasak M. 1985. Possible role for metallothionein in protection against radiation-induced oxidative stress. Kinetics and mechanism of its reaction with superoxide and hydroxyl radicals. Biochim Biophys Acta 827(1):36-44.
Uchida Y, Takio K, Titani K, Ihara Y, Tomonaga M. 1991. The growth inhibitory factor that is deficient in the Alzheimer's disease brain is a 68 amino acid metallothionein-like protein. Neuron 7(2):337-47.
Wu Z, Johnson KW, Choi Y, Ciardelli TL. 1995. Ligand binding analysis of soluble interleukin-2 receptor complexes by surface plasmon resonance. J Biol Chem 270(27):16045-51.
Anderson. J. L. B, E. F., Pickup, P. 1996. Dynamic electrochemistry: methodology and application. Anal Chem 68:379R-444R.
Bal W, Christodoulou J, Sadler PJ, Tucker A. 1998. Multi-metal binding site of serum albumin. J Inorg Biochem 70(1):33-9.
Bontidean I, Ahlqvist J, Mulchandani A, Chen W, Bae W, Mehra RK, Mortari A, Csoregi E. 2003. Novel synthetic phytochelatin-based capacitive biosensor for heavy metal ion detection. Biosens Bioelectron 18(5-6):547-53.
Carter DC, Ho JX. 1994. Structure of serum albumin. Adv Protein Chem 45:153-203.
Cominos X, Athanaselis S, Dona A, Koutselinis A. 2001. Analysis of total mercury in human tissues prepared by microwave decomposition using a hydride generator system coupled to an atomic absorption spectrometer. Forensic Sci Int 118(1):43-7.
DelRaso NJ, Foy BD, Gearhart JM, Frazier JM. 2003. Cadmium uptake kinetics in rat hepatocytes: correction for albumin binding. Toxicol Sci 72(1):19-30.
Durrieu C, Tran-Minh C. 2002. Optical algal biosensor using alkaline phosphatase for determination of heavy metals. Ecotoxicol Environ Saf 51(3):206-9.
Fisher RJ, Fivash M. 1994. Surface plasmon resonance based methods for measuring the kinetics and binding affinities of biomolecular interactions. Curr Opin Biotechnol 5(4):389-95.
Giroux E, Schoun J. 1981. Copper and zinc ion binding by bovine, dog, and rat serum albumins. J Inorg Biochem 14(4):359-62.
Giroux EL, Durieux M, Schechter PJ. 1976. A study of zinc distribution in human serum. Bioinorg Chem 5(3):211-8.
Goumakos W, Laussac JP, Sarkar B. 1991. Binding of cadmium(II) and zinc(II) to human and dog serum albumins. An equilibrium dialysis and 113Cd-NMR study. Biochem Cell Biol 69(12):809-20.
He XM, Carter DC. 1992. Atomic structure and chemistry of human serum albumin. Nature 358(6383):209-15.
Karlsson R, Falt A. 1997. Experimental design for kinetic analysis of protein-protein interactions with surface plasmon resonance biosensors. J Immunol Methods 200(1-2):121-33.
Kragh-Hansen U. 1985. Relations between high-affinity binding sites of markers for binding regions on human serum albumin. Biochem J 225(3):629-38.
Kuswandi B. 2003. Simple optical fibre biosensor based on immobilised enzyme for monitoring of trace heavy metal ions. Anal Bioanal Chem 376(7):1104-10.
Lau SJ, Sarkar B. 1971. Ternary coordination complex between human serum albumin, copper (II), and L-histidine. J Biol Chem 246(19):5938-43.
Laurie SH, Pratt DE. 1986. Copper-albumin: what is its functional role? Biochem Biophys Res Commun 135(3):1064-8.
Laussac JP, Sarkar B. 1984. Characterization of the copper(II)- and nickel(II)-transport site of human serum albumin. Studies of copper(II) and nickel(II) binding to peptide 1-24 of human serum albumin by 13C and 1H NMR spectroscopy. Biochemistry 23(12):2832-8.
Lopez-Artiguez M, Camean A, Repetto M. 1993. Preconcentration of heavy metals in urine and quantification by inductively coupled plasma atomic emission spectrometry. J Anal Toxicol 17(1):18-22.
Lu Y, Liu J, Li J, Bruesehoff PJ, Pavot CM, Brown AK. 2003. New highly sensitive and selective catalytic DNA biosensors for metal ions. Biosens Bioelectron 18(5-6):529-40.
Masuoka J, Hegenauer J, Van Dyke BR, Saltman P. 1993. Intrinsic stoichiometric equilibrium constants for the binding of zinc(II) and copper(II) to the high affinity site of serum albumin. J Biol Chem 268(29):21533-7.
Menegario AA, Packer AP, Gine MF. 2001. Determination of Ba, Cd, Cu, Pb and Zn in saliva by isotope dilution direct injection inductively coupled plasma mass spectrometry. Analyst 126(8):1363-6.
Mernagh DR, Janscak P, Firman K, Kneale GG. 1998. Protein-protein and protein-DNA interactions in the type I restriction endonuclease R.EcoR124I. Biol Chem 379(4-5):497-503.
Palumaa P, Vasak M. 1992. Binding of inorganic phosphate to the cadmium-induced dimeric form of metallothionein from rabbit liver. Eur J Biochem 205(3):1131-5.
Peters T, Jr. 1985. Serum albumin. Adv Protein Chem 37:161-245.
Ramanathan S, Ensor M, Daunert S. 1997. Bacterial biosensors for monitoring toxic metals. Trends Biotechnol 15(12):500-6.
Sadler PJ, Tucker A, Viles JH. 1994. Involvement of a lysine residue in the N-terminal Ni2+ and Cu2+ binding site of serum albumins. Comparison with Co2+, Cd2+ and Al3+. Eur J Biochem 220(1):193-200.
Sadler PJ, Viles JH. 1996. 1H and (113)Cd NMR Investigations of Cd(2+) and Zn(2+) Binding Sites on Serum Albumin: Competition with Ca(2+), Ni(2+), Cu(2+), and Zn(2+). Inorg Chem 35(15):4490-4496.
Scott BJ, Bradwell AR. 1983. Identification of the serum binding proteins for iron, zinc, cadmium, nickel, and calcium. Clin Chem 29(4):629-33.
Soares ME, Bastos ML, Ferreira M. 2000. Selective determination of chromium (VI) in powdered milk infant formulas by electrothermal atomization atomic absorption spectrometry after ion exchange. J AOAC Int 83(1):220-3.
Sokolowska M, Krezel A, Dyba M, Szewczuk Z, Bal W. 2002. Short peptides are not reliable models of thermodynamic and kinetic properties of the N-terminal metal binding site in serum albumin. Eur J Biochem 269(4):1323-31.
Stewart AJ, Blindauer CA, Berezenko S, Sleep D, Sadler PJ. 2003. Interdomain zinc site on human albumin. Proc Natl Acad Sci U S A 100(7):3701-6.
Sudlow G, Birkett DJ, Wade DN. 1975. The characterization of two specific drug binding sites on human serum albumin. Mol Pharmacol 11(6):824-32.
Sugio S, Kashima A, Mochizuki S, Noda M, Kobayashi K. 1999. Crystal structure of human serum albumin at 2.5 A resolution. Protein Eng 12(6):439-46.
Tsalev DL. 1984. Atomic Absorption Spectrometry in Occupational and environmental Health practice.
Watkins SR, Hodge RM, Cowman DC, Wickham PP. 1977. Cadmium-binding serum protein. Biochem Biophys Res Commun 74(4):1403-10.
Wu Z, Johnson KW, Choi Y, Ciardelli TL. 1995. Ligand binding analysis of soluble interleukin-2 receptor complexes by surface plasmon resonance. J Biol Chem 270(27):16045-51.